Open AccessAccurate and rapid detection of heterozygous carriers of a deletion by combined polymerase chain reaction and high-performance liquid chromatography.
Author(s) -
Junichi Asakawa,
Chiyoko Satoh,
Yosuke Yamasaki,
Shi-Han Chen
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.19.9126
Subject(s) - polymerase chain reaction , microbiology and biotechnology , heterozygote advantage , dna , genomic dna , polymerase , biology , duchenne muscular dystrophy , chromatography , chemistry , genetics , gene , genotype
We have developed a technique to detect accurately heterozygous carriers of a deletion. Specific target sequences were amplified by the polymerase chain reaction (PCR), and the products subsequently were analyzed by high-performance liquid chromatography. Examples from four loci demonstrated that 24-27 cycles of amplification for a single-copy DNA, based on 50 ng of genomic DNA, results in excellent quantitation that readily permits the detection of heterozygous carriers of a deletion. We have demonstrated that triplex PCR (three targets in a single PCR) entails no loss of precision. We also have demonstrated that this method can accurately differentiate the heterozygous carriers of a deletion from normal individuals in four family studies, three for Duchenne muscular dystrophy patients and one for a hemophilia B patient.